Process for producing microcapsule

ABSTRACT

A polyelectrolyte solution as a disperse phase is fed into one of the chambers which are partitioned by a plate having a plurality of narrow holes (microchannels), a continuous phase is fed into the other chamber, and pressure is applied to the disperse phase forcing it through the holes into the continuous phase so as to prepare an emulsion. This emulsion is demulsified, and at the same time the disperse phase is brought into contact with a polyelectrolyte solution having a reverse electric charge to the disperse phase or a polyvalent ion solution, and a gel layer is formed around the spherical disperse phase by a polyelectrolyte reaction. Thereby, a double-structured capsule is obtained, in which the outside is insoluble gel and the inside is a polyelectrolyte solution to which a cell has been added.

TECHNICAL FIELD

The present invention relates to a manufacturing method formicrocapsules which are used in a DDS (drug delivery system), a foodindustry, or cosmetic manufacturing.

BACKGROUND ART

As a capsule to be transplanted into a body, there has been known amicrocapsule of 500-800 μm in which one or two cell(s) (islands ofLangerhans) is encapsulated. (Document, “protein, nucleic acid, enzymeVol. 45, No. 13” 2000)

In this capsule, an outside hydrogel functions as a barrier to an attackfrom an immune mechanism (rejection reaction), and thereby the islandsof Langerhans can secrete insulin for a long period of time in the body.

The first proposal regarding such a capsule was made in U.S. Pat. No.4,352,883 (1979). This known art material describes that a cell is fixedin calcium alginate gel.

As a technique for fixing a cell inside a shell which endures an attackfrom an immune mechanism and transplanting into a body, there have alsobeen known Japanese Patent Application Publication No. 10-500889,Japanese Patent Application Publication No. 11-130698, and JapanesePatent Application Publication No. 2002-507473.

Japanese Patent Application Publication No. 10-500889 has disclosed thata rotavirus is encapsulated in a microcapsule, the outside shell ofwhich is made by a reaction of alginic acid and spermine, and the insideof which is an aqueous core.

Japanese Patent Application Publication No. 11-130698 has disclosed thatan alginic acid aqueous solution (W) is emulsified in fatty acid ester(O) so as to produce a W/O emulsion, polyvalent metal (Ca²⁺ or Ba²⁺) isadded to the emulsion so as to form primary particles of alginic acidpolyvalent metal salt (gel) having a diameter of 0.01-5 μm, and a poorlysoluble medicine is carried by the aggregate of the primary particles.

Japanese Patent Application Publication No. 2002-507473 has disclosedthat particles of an alginic acid aqueous solution are prepared byatomizing, and microcapsules of 100-400 μm are obtained by allowing theparticles of an alginic acid aqueous solution to collide with a Ca²⁺solution flowing down in a film shape.

In addition, Japanese Patent Application Publication No. 09-500132 hasproposed a vaccine having a size of 15 μm or less for oral delivery inwhich a hydrogel is used to encapsulate.

The above-mentioned outside shell (gel) is formed by a polyelectrolytereaction. Specifically, a poly anion solution such as an alginic acidsolution is dropped onto a poly cation solution by using a nozzle asdisclosed in “Biotechnology Progress 13, 562-568, 1997”.

Also, a method using a double nozzle in order to reduce the diameter ofa capsule has been disclosed in “AICHE J, 40, 1026-1031, 1994”. In thismethod, a capsule of 2 mm-200 μm is prepared by feeding apolyelectrolyte solution from an inner nozzle and feeding air from anouter nozzle.

According to the above-mentioned conventional methods, it is possible toobtain microcapsules having a diameter in the range of from 0.01 μm toseveral hundreds of μm. However, in the conventional methods, thedistribution of the particle diameter is wide, that is, it is difficultto obtain microcapsules having a uniform diameter.

Specifically, in Japanese Patent Application Publication No. 10-500889,and Japanese Patent Application Publication No. 2002-507473, an alginicacid solution is atomized into the air so as to make small particles,and then the particles are brought into contact with a Ca²⁺ aqueoussolution. However, in such methods, capsules having a uniform diametercannot be obtained.

In Japanese Patent Application Publication No. 11-130698, a W/O emulsionis produced by a conventional method, and this emulsion is brought intocontact with a Ca²⁺ aqueous solution. In this case, however, it isdifficult to control the diameter of the droplets of the disperse phasewithin a certain range. Accordingly, although a very fine particle canbe produced, it is impossible to produce a capsule having a doublestructure in which an aqueous solution is encapsulated inside and theoutside shell is gel.

The above-mentioned documents suggest that a microcapsule encapsulatinga cell can be transplanted in a body so as to function as “a micromedicine factory”. For this purpose, the cell needs to not only secretean effective material such as insulin or an antineoplastic agent butalso be alive in the microcapsule for a long period of time.

In order to allow the cell to be alive in the microcapsule for a longperiod of time, the particle diameter of the microcapsule is animportant factor.

Specifically, in the microcapsule for encapsulating a cell, the outsideshell (gel) needs to not only endure an attack from an immune mechanismbut also release a secretion from the cell, take nutrition necessary forthe cell to keep alive, and excrete waste products generated in thecapsule.

According to the present inventors' research, when the radius of themicrocapsule is more than 150 μm (diameter: 300 μm), nutrition cannot befed to the cell fixed in the center, and waste products cannot beexcreted from the cell. Consequently, the cell dies. In contrast, if thediameter of the microcapsule is too small, it is impossible to fix acell inside.

Therefore, most of microcapsules must have a diameter within anextremely limited range.

As for microcapsules for encapsulating a cell, the diameter distributionmust be within a narrow range of 50-300 μm. Although a conventionalmethod in which dropping is used can manufacture a microcapsule having adiameter within the above-mentioned range, it is impossible tomanufacture microcapsules having a uniform diameter. Also, in aconventional method which uses an emulsion obtained by simple stirring,it is impossible to manufacture microcapsules having a uniform diameterwithin a certain range.

Incidentally, microcapsules having a uniform particle diameter arerequired in other fields such as food or cosmetic.

DISCLOSURE OF THE INVENTION

In order to solve the above-mentioned problems, according to the presentinvention, there is provided a manufacturing method for microcapsulescomprising the steps of preparing an emulsion which contains apolyelectrolyte solution as a disperse phase having a uniform diameter,demulsifying the emulsion, and contacting the polyelectrolyte solutionas a disperse phase with a polyelectrolyte solution having a reverseelectric charge to the polyelectrolyte solution as a disperse phase or apolyvalent ion solution at the same time of the demulsifying step so asto form a gel layer made of a polyelectrolyte complex around fineparticles of the polyelectrolyte solution as a disperse phase by apolyelectrolyte reaction.

In the present invention, a polyelectrolyte solution is turned into anemulsion which contains a disperse phase having a uniform diameterwithout directly contacting the polyelectrolyte solution with anotherpolyelectrolyte solution having a reverse electric charge thereto or apolyvalent ion solution, and thereafter the emulsion is brought intocontact with a polyelectrolyte solution having a reverse electric chargeor a polyvalent ion solution. As a result of this, it is possible toobtain microcapsules having substantially the same diameter as thedisperse phase.

In order to obtain microcapsules having a uniform diameter, it isnecessary to obtain an emulsion, the disperse phase of which has auniform diameter. For this purpose, preferably, the disperse phase andthe continuous phase are separated by a plate having penetrating holes,and the disperse phase is pushed into the continuous phase asmicrospheres by applying greater pressure to the disperse phase than tothe continuous phase.

Also, in order to contact the disperse phase with a polyelectrolytesolution having a reverse electric charge or a polyvalent ion solutionefficiently, it is necessary to demulsify the emulsion. There are twomethods for demulsifying. The first one is a method in which theconcentration of a surface-active agent, which is commonly added to acontinuous phase to keep the emulsion state, is reduced by adding thesame material as the continuous phase (such as hexane) or a solublematerial to the continuous phase. The second one is a method in which asurface-active agent is originally not added at the time of preparingthe emulsion. In the second method, since the emulsion is demulsified ina short period of time, the contacting step must be conductedimmediately.

Examples of the disperse phase include an alginic acid, carboxymethylcellulose, pectin, carrageenan, sulfate cellulose, and chondroitinsulfuric acid. Examples of the polyelectrolyte to be reacted with thedisperse phase include a polyamino acid (such as polyhistidine,polylysine, or polyornithine), polymer containing a primary amine group,a secondary amine group, a tertiary amine group, or pyridinyl nitrogen(such as polyethylene imine, polyallyl imine, polyether amine, orpolyvinyl pyridine), and aminated polysaccharide (such as chitosan).Examples of the polyvalent ion to be reacted with the disperse phaseinclude Ca²⁺, Ba²⁺, Pb²⁺, Cu²⁺, Cd²⁺, Sr²⁺, Co²⁺, Ni²⁺, Zn²⁺ and Mn²⁺.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1(a)-(c) show an emulsion preparing step of a manufacturing methodfor microcapsules according to the present invention;

FIGS. 2(a) and (b) show manufacturing of microcapsules according to thepresent invention;

FIG. 3 is an enlarged cross-sectional view of a microcapsule obtained bythe method according to the present invention;

FIG. 4 is a cross-sectional view of an apparatus for preparing anemulsion which is used in Examples 1 and 2;

FIG. 5 is a photomicrograph showing a state of preparing an emulsion inExample 1;

FIG. 6 is a photomicrograph of a microcapsule obtained in Example 1,

FIG. 7 is a photomicrograph showing a state of preparing an emulsion inExample 2; and

FIG. 8 is a photomicrograph of a microcapsule obtained in Example 2,

BEST MODE FOR CARRYING OUT THE INVENTION

Embodiments of the present invention will now be described withreference to the attached drawings. FIGS. 1(a)-(c) show an emulsionpreparing step of a manufacturing method for microcapsules according tothe present invention, FIGS. 2(a) and (b) show manufacturing ofmicrocapsules according to the present invention, and FIG. 3 is anenlarged cross-sectional view of a microcapsule obtained by the methodaccording to the present invention.

As shown in FIG. 1(a), a polyelectrolyte solution as a disperse phase isfed into one of the chambers which are partitioned by a plate having aplurality of narrow holes, and a continuous phase (hexane) is fed intothe other chamber.

Next, pressure is applied to the polyelectrolyte solution. Then, thepolyelectrolyte solution enters the continuous phase while turning intoa disperse phase as shown in FIG. 1(b), and an emulsion is prepared asshown in FIG. 1(c).

The shape of the disperse phase is spherical. The diameter of thespherical disperse phase depends on the size of the holes. If the sizeof the holes is uniform, the diameter of the obtained disperse phasebecomes uniform. The holes are formed by plasma etching which is usedfor manufacturing an integrated circuit. In addition, a more uniformdisperse phase can be obtained by making the shape of the holenon-circular.

The emulsion prepared in the above-mentioned manner is put on apolyelectrolyte solution having a reverse electric charge to thedisperse phase or a polyvalent ion solution within a single vessel in astate of keeping the phase separation as shown in FIG. 2(a), andthereafter the emulsion is demulsified.

The emulsion is demulsified by adding the same material as thecontinuous phase (hexane) or a soluble material to the continuous phase(such as soybean oil, triolein, or octane) to the emulsion so as toreduce the concentration of the surface-active agent in the continuousphase, or by originally not adding a surface-active agent to thecontinuous phase.

When the emulsion has been demulsified, the disperse phase is contactedand reacted with the polyelectrolyte solution having a reverse electriccharge to the disperse phase or the polyvalent ion solution, and a gellayer is formed around the spherical disperse phase. Finally, as shownin FIG. 3, a double-structured capsule is obtained, in which the outsideis insoluble gel and the inside is a polyelectrolyte solution to which acell has been added.

The microcapsule encapsulating a cell can be used for a medicaltreatment of a human body or a prevention against disease. In such acase, the microcapsule is injected into the parts of a human body by aninjector, a catheter or an operation.

Next, embodiments of the present invention will be explained. FIG. 4 isa cross-sectional view of an apparatus for preparing an emulsion whichis used in Examples 1 and 2. The apparatus for preparing an emulsion iscomprised of an annular case 1, and plates 2, 3, 4 and spacers which areassembled within the case 1. The disperse phase flows through aliquid-sealed first passage 11, and the continuous phase and theemulsion flows through a liquid-sealed second passage 12. The firstpassage 11 and the second passage 12 are connected by narrow holes(microchannels) which are provided in the intermediate plate 3. P1 is afeeding pump for the disperse phase, P2 is a feeding pump for thecontinuous phase, and P3 is a withdrawing pump for the emulsion. Atransparent window 13 and a CCD camera are also provided in theapparatus.

EXAMPLE 1

Chitosan (manufactured by KIMICA Corporation) and sodium carboxymethylcellulose (manufactured by Nippon Rika Co., Ltd.) were employed as a rawmaterial of the capsule. Hexane was used as a continuous phase, andTGCR-310 (manufactured by Sakamoto Yakuhin Kogyo Co., Ltd.) was used asa surface-active agent.

Carboxymethyl cellulose of 0.8 wt % was prepared, supplied to the firstpassage 11 by using the pump P1, and pushed into hexane flowing throughthe second passage 12 via the holes of the intermediate plate 3, so asto prepare a monodisperse W/O emulsion. FIG. 5 is a photomicrographshowing an enlarged view of this W/O emulsion.

This emulsion and a chitosan solution of 0.5 wt % (solvent: acetic acid)were put into a single vessel in a state of keeping the phaseseparation, and hexane was added to the emulsion.

By adding hexane, the emulsion was demulsified due to a decrease in theconcentration of the surface-active agent. The carboxymethyl celluloseand the chitosan solution were brought into contact with respect to eachother immediately, and polyelectrolyte complex gel was formed around thecarboxymethyl cellulose droplets, so as to manufacture microcapsules ofchitosan and carboxymethyl cellulose.

As mentioned above, by using the narrow holes (microchannels) formed inthe plate (division wall), a monodisperse emulsion having a particlediameter of about 50 μm could be prepared. The capsules made from theemulsion were also monodisperse, that is, the diameter of the capsuleshad substantially the same particle diameter.

The preparation of the manufactured microcapsules was observed by amicroscope, and the state where the surface film of the capsule wascomprised of countless gel fibers was observed as shown in FIG. 6.

EXAMPLE 2

An alginic acid (manufactured by KIMICA Corporation) was used as a rawmaterial of the capsule. Soybean oil was used for an oil phase. Anaqueous solution including a 0.1 M calcium chloride solution was usedfor a reaction solution.

An aqueous solution of an alginic acid of 1.5% (disperse phase) wassupplied to the first passage 11, and soybean oil (continuous phase) inwhich no surface-active agent was added was supplied to the secondpassage 12. The aqueous solution of an alginic acid was pushed into thesoybean oil via the holes (microchannels), so as to prepare an emulsion.

This emulsion was brought into contact with an aqueous solution ofcalcium chloride (polyvalent ion). As a result of this, capsules ofcalcium alginate were obtained.

According to Example 2, as shown in FIG. 7, the obtained emulsion washomogenous, and the particle diameter of the disperse phase (droplet)was about 80 μm. This emulsion was contacted with the aqueous solutionof calcium chloride, and thereby capsules having a particle diameter ofaround 100 μm were obtained.

In the apparatus used in the above-mentioned examples, after theemulsion was prepared, the disperse phase of the emulsion and apolyelectrolyte solution having a reverse electric charge or apolyvalent ion solution were contacted with respect to each other withinanother vessel so as to manufacture microcapsules. However, it is alsopossible to manufacture microcapsules in a single apparatus.

For example, a division wall may be provided in a substantially centralarea of the first passage 11 to divide the first passage into left andright sections. In this case, a disperse phase is supplied to the leftsection of the first passage by the pump P1 in the same manner as usual,and a polyelectrolyte solution having a reverse electric charge or apolyvalent ion solution is supplied to the right section of the firstpassage by another pump. With this, an emulsion is manufactured in anarea on the upstream side of the second passage 12 where the dispersephase is supplied via the holes of the plate 3, and microcapsules aremanufactured in an area on the downstream side (the right side of thedrawing) where a polyelectrolyte solution having a reverse electriccharge or a polyvalent ion solution is supplied via the holes of theplate 3.

In the above-mentioned method in which the disperse phase is introducedinto the continuous phase via the narrow holes penetrating the thicknessdirection of the plate 3, the particle diameter of the disperse phaseparticles (microcapsules) in the emulsion depends on the diameter of theholes, and it is difficult to adjust the particle diameter.

In order to overcome this problem, there is another way to manufacturean emulsion which does not use narrow holes. Specifically, by allowing acontinuous phase to flow through a microchannel, and a disperse phase toflow through another microchannel, both of which join with each other,the continuous phase and the disperse phase are allowed to join in astate of a laminar flow, and thereafter the flow rate of the continuousphase and the disperse phase are reduced in a dramatic way, so that thedisperse phase particles can appear in the continuous phase. In thiscase, the disperse phase is taken into the continuous phase per oneparticle by a shearing stress, and the particle diameter can becontrolled by adjusting the flow rate of the continuous phase and thedisperse phase.

The microchannels are formed on a glass base or a silicon base. As ameans for allowing the continuous phase and the disperse phase to join,the passages of the continuous phase may be arranged to join with thepassage of the disperse phase from the both sides at an angle of 30-80°.Also, as a means for reducing the flow rate in a dramatic way, a poolhaving a large volume of capacity may be provided.

As mentioned above, according to the present invention, it is possibleto stably produce a large quantity of capsules having a double structurein which a polyelectrolyte solution is encapsulated within a gel layerformed by a reaction between this polyelectrolyte solution and anotherpolyelectrolyte solution in a state where the particle diameter is keptuniform.

Consequently, it is possible to obtain an effective capsule in a medicalfield such as for encapsulating a cell as well as in a food or cosmeticfield.

INDUSTRIAL APPLICABILITY

The present invention can effectively be used in a DDS (drug deliverysystem), a medical treatment for a human body, a food industry, orcosmetic manufacturing.

1-8. (canceled)
 9. A manufacturing method for microcapsules comprisingthe steps of: preparing an emulsion which contains a polyelectrolytesolution as a disperse phase having a uniform diameter according to themethod of claim 19; demulsifying the emulsion; and contacting thepolyelectrolyte solution as a disperse phase with a polyelectrolytesolution having a reverse electric charge to the polyelectrolytesolution as a disperse phase or a polyvalent ion solution at the sametime as the demulsifying step so as to form a gel layer made of apolyelectrolyte complex around fine particles of the polyelectrolytesolution as a disperse phase by a polyelectrolyte reaction.
 10. Themanufacturing method for microcapsules according to claim 9, wherein themicrochannels are formed on a glass base or a silicon base.
 11. Themanufacturing method for microcapsules according to claim 9, wherein theflow rate is reduced in a dramatic way by flowing the joined continuousand disperse phases into a pool having a large volume of capacity.
 12. Amanufacturing method for microcapsules, which is performed in a singleapparatus comprising a case, a first passage for a disperse phase, asecond passage for a continuous phase, a plate positioned between thefirst passage and the second passage, penetrating holes formed in theplate, and a division wall provided in a substantially central area ofthe first passage to divide the first passage into first and secondsections, comprising the steps of: supplying a continuous phase to thesecond passage; supplying a polyelectrolyte solution as a disperse phaseto the first section of the first passage in a state of applying greaterpressure to the polyelectrolyte solution than to the continuous phase soas to push the disperse phase into the continuous phase via thepenetrating holes to prepare an emulsion; supplying a polyelectrolytesolution having a reverse electric charge to that of the polyelectrolytesolution as a disperse phase or a polyvalent ion solution to the secondsection of the first passage in a state of applying greater pressure tothe polyelectrolyte solution having a reverse electric charge or thepolyvalent ion solution than to the continuous phase; and contacting thepolyelectrolyte solution as a disperse phase with the polyelectrolytesolution having a reverse electric charge or the polyvalent ion solutionwhile the emulsion is demulsified so as to form a gel layer made of apolyelectrolyte complex around fine particles of the polyelectrolytesolution as a disperse phase by a polyelectrolyte reaction.
 13. Themanufacturing method for microcapsules according to claim 9, wherein theemulsion is demulsified by adding the same material as the continuousphase or a material which is soluble in the continuous phase to theemulsion so as to reduce the concentration of a surface-active agent inthe emulsion.
 14. The manufacturing method for microcapsules accordingto claim 9, wherein the emulsion does not contain a surface-active agentand the emulsion is demulsified by being contacted with thepolyelectrolyte solution having a reverse electric charge or thepolyvalent ion solution.
 15. The manufacturing method for microcapsulesaccording to claim 9, wherein the disperse phase is selected from agroup consisting of an alginic acid, carboxymethyl cellulose, pectin,carrageenan, sulfate cellulose, and chondroitin sulfuric acid; thepolyelectrolyte to be reacted with the disperse phase is selected from agroup consisting of a polyamino acid, polymer containing a primary aminegroup, a secondary amine group, a tertiary amine group, or pyridinylnitrogen, and aminated polysaccharide; and the polyvalent ion in thepolyvalent ion solution is selected from a group consisting of Ca²⁺,Ba²⁺, Pb²⁺, Cu²⁺, Cd²⁺, Sr²⁺, Co²⁺, Ni²⁺ and Mn²⁺.
 16. The manufacturingmethod for microcapsules according to claim 9, wherein a cell whichgenerates a desired material is added to the polyelectrolyte solution asa disperse phase in advance of the emulsion preparation step.
 17. Themanufacturing method for microcapsules according to claim 9, wherein thediameter of the disperse phase is within the range of 50-300 μm.
 18. Amethod for treating a human body, wherein the microcapsule manufacturedby the method according to claim 9 is injected into parts of a humanbody by an injector, a catheter or an operation.
 19. A method forpreparing an emulsion comprising the steps of: allowing a continuousphase material to flow through a microchannel; allowing apolyelectrolyte solution as a disperse phase to flow through anothermicrochannel, the microchannels being joined with each other to allowthe continuous phase and the disperse phase to join in a state of alaminar flow; and thereafter reducing the flow rate of the continuousphase and the disperse phase in a dramatic way so as to prepare anemulsion which contains the polyelectrolyte solution as a disperse phasehaving a uniform diameter.
 20. A manufacturing method for microcapsulescomprising the steps of: preparing an emulsion which contains apolyelectrolyte solution as a disperse phase having a uniform diameterand a continuous phase; demulsifying the emulsion; and contacting thepolyelectrolyte solution as a disperse phase with a polyelectrolytesolution having a reverse electric charge to the polyelectrolytesolution as a disperse phase or a polyvalent ion solution at the sametime as the demulsifying step so as to form a gel layer made of apolyelectrolyte complex around fine particles of the polyelectrolytesolution as a disperse phase by a polyelectrolyte reaction.
 21. Themanufacturing method for microcapsules according to claim 20, whereinthe emulsion is prepared by separately feeding the disperse phase andthe continuous phase with a plate having penetrating holes, and applyinggreater pressure to the disperse phase than to the continuous phase soas to push the disperse phase into the continuous phase as microspheres.22. The manufacturing method for microcapsules according to claim 20,wherein the emulsion is demulsified by adding the same material as thecontinuous phase or a material which is soluble in the continuous phaseto the emulsion so as to reduce the concentration of a surface-activeagent in the emulsion.
 23. The manufacturing method for microcapsulesaccording to claim 20, wherein the emulsion does not contain asurface-active agent, and the emulsion is demulsified by being contactedwith the polyelectrolyte solution having a reverse electric charge tothe polyelectrolyte solution as a disperse phase or the polyvalent ionsolution.
 24. The manufacturing method for microcapsules according toclaim 20, wherein the disperse phase is selected from a group consistingof an alginic acid, carboxymethyl cellulose, pectin, carrageenan,sulfate cellulose, and chondroitin sulfuric acid; the polyelectrolyte tobe reacted with the disperse phase is selected from a group consistingof a polyamino acid, polymer containing a primary amine group, asecondary amine group, a tertiary amine group, or pyridinyl nitrogen,and aminated polysaccharide; and the polyvalent ion of the polyvalention solution is selected from a group consisting of Ca²⁺, Ba²⁺, Pb²⁺,Cu²⁺, Cd²⁺, Sr²⁺, Co²⁺, Ni²⁺ and Mn²⁺.
 25. The manufacturing method formicrocapsules according to claim 20, wherein a cell which generates adesired material is added to the polyelectrolyte solution as a dispersephase in advance of said emulsion preparation step.
 26. Themanufacturing method for microcapsules according to claim 20, whereinthe diameter of the disperse phase is within the range of 50-300 μm. 27.A method for treating a human body, wherein the microcapsulemanufactured by the method according to claim 20 is injected into partsof a human body by an injector, a catheter or an operation.